Molecular cloning is an essential toolset in the study of genetic material in laboratory settings. Through the usage of recombinant polymerases, restriction enzymes and ligases, genetic material can be isolated, amplified and inserted into DNA vectors for downstream functional characterization. In addition to isolating and studying naturally existing genetic material, synthetic recombinant DNA can be created and characterized by incorporating synthetic oligonucleotides into a molecular cloning workflow.
Briefly, at any given position in any gene, there are four possible nucleotides: adenine (A), guanine (G), cytosine (C) and thymine (T). Triplets of these nucleotides in different combinations are known as codons. Each codon in turn encodes for one of the twenty amino acids required for protein synthesis. There may be more than one triplet combination of nucleotides which encode for any given amino acid. Therefore, alteration of, or in other words, a mutant triplet of DNA may still result in the correct amino acid being placed at the correct position in a protein. This concept is referred to as degeneracy of codons. Alternatively, a degenerate codon synthesized in the context of a short oligonucleotide refers to a mixture of A, C, G or T nucleotides at any position and this tool may be used to introduce genetic diversity at any position. Genetically diverse libraries are typically encoded using NNK or NNS codons, where N=A, G, C or T, K=G or T, and S=C or G different amino acids.
Single-site saturation mutagenesis is a process whereby an individual codon is altered or mutated to any other combinations of A, C, G or T. This process is typically conducted using enzyme-based biochemical reactions or de novo chemical synthesis. With regards to enzyme-based biochemical reactions, a researcher will utilize a degenerate oligonucleotide DNA primer in a polymerase chain reaction (PCR). With this enzyme-based PCR mutagenesis technique, a single set of site-directed mutagenesis oligonucleotides is employed, thus resulting in the enzyme-based PCR mutagenesis technique being limited to a single nucleotide triplet site to be targeted for a given gene. The newly amplified DNA strands will incorporate the genetic diversity encoded by the degenerate codon into the PCR products. With regards to de novo chemical synthesis, a researcher will synthesize new strands of DNA up to the location where saturation mutagenesis is required, at which point they would add in a mixture of A, C, G and T nucleotides to create genetic diversity at the targeted location, having one nucleotide incorporated next in the sequence. By adding in a mixture of nucleotides, genetic diversity is created at that targeted location in newly synthesized DNA strands. Similar to the enzyme-based PCR mutagenesis technique, using de novo chemical synthesis, only a single nucleotide is targetable at once since only a single strand of DNA can be synthesized in a single chemical reaction. Therefore, a major drawback of both the enzyme reaction method and de novo synthesis is that a unique PCR reaction or DNA synthesis reaction is required for every codon that requires saturation mutagenesis thus being limited to targeting a single nucleotide site. Each process must be repeated for each nucleotide base in a gene requiring substitution so as to create a genetically diverse DNA library.
Accordingly, with current and commercially available methods, the single site saturation mutagenesis techniques briefly noted above only facilitate saturation mutagenesis at a single triplet site. Applying a single site saturation mutagenesis technique approach to mutate several DNA positions in a gene is both time consuming and cost ineffective and scaling up these processes to mutagenize multiple codons in a saturated fashion is therefore generally infeasible for both technologies. Consequently, development of a high throughput approach for multi-site saturation DNA mutagenesis would be desirable to facilitate a high throughput method.
Using currently available techniques, the in vitro generation of several mutants using multi-site saturation DNA mutagenesis of a given gene or DNA molecule is not possible in a high throughput manner. Thus, generation of a mutant DNA library, whereby multiple codon sites are mutated for a given gene is cumbersome and time consuming using the currently known and available single-site saturation mutagenesis techniques.
It would be desirable to create a streamlined and cost-effective process for mutagenizing multiple codons in a saturated manner which would serve as an efficient tool for researchers and enable the study of, for example, the consequences of variation or mutation on gene function, human health and drug sensitivity.
This background information is provided to reveal information believed by the applicant to be of possible relevance. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art.